Abstract Epigenetic therapy aims to reprogram gene expression in cancer cells to achieve a therapeutic effect. To date, DNMT inhibition is the most effective form of epigenetic therapy in myeloid leukemias. We have developed and validated a live cell assay to screen for drugs that achieve the same degree of epigenetic reprogramming as DNMT inhibition. Using this screen, we discovered a new class of epigenetic drugs that activate silenced expression through inhibition of CDK9. CDK9 is a transcriptional regulator previously linked to gene activation through the pTEFb complex that phosphorylates RNAPII and promotes transcriptional elongation. Our new data now place CDK9 at the heart of a node that regulates both gene silencing and activation in proliferating cells. As such, targeting CDK9 has pleotropic effects on gene expression that appear ideal from an anti-tumor perspective: One observes simultaneous gene activation (of tumor suppressors), repression (of oncogenes), and induction of an interferon immune signature, which may be immune-sensitizing. Known CDK9 inhibitors (flavopiridol, SNS-032) have activity in leukemias but are marred by serious chemotherapy-like toxicities. Examining published data, we find that doses of these drugs in use clinically are at least an order of magnitude higher than what is needed to inhibit CDK9, and we speculate that the toxicity observed is typical of cross-target inhibition of other CDKs (e.g. CDK1/2). Thus, we hypothesize that low doses of CDK9-selective drugs may preserve activity through epigenetic effects of CDK9 inhibition, while reducing toxicity by avoiding other CDKs. In this grant, we will elucidate mechanisms of epigenetic effects of CDK9, determine the downstream effects of CDK9 inhibition on cellular function and immune responses, and conduct a clinical trial of a new CDK9- selective drug in myeloid leukemias. Successful completion of these aims will introduce a new form of epigenetic therapy in the treatment of leukemias.